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Ngs part ii 2013
This document discusses next-generation sequencing (NGS) and its role in cancer research. It begins with an introduction to NGS technology and applications. The speaker then discusses how NGS can help with cancer research by identifying new cancer genes and mutations, assisting with diagnosis and targeted therapies, and enabling patient stratification. The need for NGS is explained by discussing cancer genetics, statistics on cancer incidence, and the need to identify driver mutations and develop personalized treatments. Qiagen offers an end-to-end NGS workflow and solutions including sample preparation, target enrichment, library preparation, sequencing, and data analysis. Their targeted gene panels and analysis software provide a streamlined sample-to-result experience for cancer research.
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Ngs part ii 2013
- 1. Sample & AssayTechnologies Next Generation Sequencing for Cancer Research Vikram Devgan, Ph.D., MBA Director, R&D vikram.devgan@qiagen.com
- 2. Sample & AssayTechnologies Welcome to the three-part webinar series Next Generation Sequencing and its role in cancer biology Webinar 1: Next-generation sequencing, an introduction to technology and applications Speaker: Quan Peng, Ph.D. Webinar 2: Speaker: Next-generation sequencing for cancer research Vikram Devgan, Ph.D., MBA Webinar 3: Speaker: Next-generation sequencing data analysis for genetic profiling Ravi Vijaya Satya, Ph.D. 2
- 3. Sample & AssayTechnologies Agenda 1. Introduction to Next Generation Sequencing in Cancer Research 2. Need for change 3. QIAGEN solutions: sample-to-result experience 3
- 4. Sample & AssayTechnologies Cancer: A Disease of Genome A C T G Variation 4
- 5. Sample & AssayTechnologies Genetic Variations AGCTCGTTGCTCAGCTC Reference genome AGCTCGTTGCTCAGCGTTC Insertion ---GCTCAGCTC AGCTC Mutations Indels Deletion Copy number variation Challenge in Treating Cancer: Structural variation Every tumor is different Every cancer patient is different 5
- 6. Sample & AssayTechnologies Cancer: Facts In 2008 over 12.7 millions new cases were diagnosed across the planet and approximately 7.6 million cancer death occurred In 2030, these numbers will rise to an expected 21.4 millions new cases and 13.2 million cancer death……….. If our ability to prevent, diagnose and treat cancer doesn't improve 6
- 7. Sample & AssayTechnologies Need of Cancer Research Identify new cancer susceptibility genes Identify mutations that drive cancer progression Assist in developing new diagnostic tests and targeted therapies Science 339, 1546 (2013); Bert Vogelstein et al. Cancer Genome Landscapes 7
- 8. Sample & AssayTechnologies e.g. lung cancer Need: Patient stratification / Personalized medicine e.g. lung cancer
- 9. Sample & AssayTechnologies Unknown 37% Current lung cancer biomarker landscape KRAS 25% EGFR (L858R) + KRAS (G12C) EGFR 23% NRAS 0.2% MAP2K1 0.4% ERBB2 1% MET 2% BRAF EML4-ALK PIKC3A 3% 6% 3% Response rates of >70% in patients with non-small cell lung cancer treated with either erlotinib or gefitinib EML4-ALK Poor response rate in patients with non-small cell lung cancer treated with either erlotinib or gefitinib Crizotinib Multiple mutation tests If tested sequentially – Not adequate tissue sample – Takes time to rule out individual mutations Need tool to conduct simultaneous genetic testing for multiple oncogenic drivers
- 10. Sample & AssayTechnologies Need: Public health Population screening for high risk preventable disorders ~0.25% of US women (375,000) carry a mutation in BRCA1/2 At very high risk of breast and ovarian cancer – 85% lifetime breast cancer risk – 25-50% lifetime ovarian cancer Knowledge of risk allows prevention
- 11. Sample & AssayTechnologies Basic Research Need of Cancer Research Clinical Research Cancer susceptibility genes Tumor sub-typing Causative mutations for multigenic diseases Biomarker identification Resistance marker identification Public Health Population screening for high risk preventable disorders Personalized treatment Therapeutic monitoring Patient stratification for clinical trails Need a tool to detect multiple mutations in multiple genes with multiple samples
- 12. Sample & AssayTechnologies Evolution of technologies for mutation detection Few ARMS PCR Liquid bead array Sequenom MALDI-TOF Next Generation Sequencing Many Next Generation Sequencing: Multiple Mutation/Multiple Genes/Multiple Samples 12
- 13. Sample & AssayTechnologies NGS: the best method for mutation detection High throughput Test many genes at once Cost effective Drastic decrease in cost of sequencing Systematic and unbiased Detection of all mutation types Quantitative Easy to quantify mutation frequency 13
- 14. Sample & AssayTechnologies Challenges in translating NGS to oncogenomics Quantity Limiting for biopsy specimens Purity (genetic heterogeneity) Cancerous cells may be a minor fraction of total sample Multiple sub-clones of cancer may be present in one tumor sample Genomic alterations in cancer found at low-frequency Data analysis Biologically interpretable data Fragmented Workflow Technologies are not jointly developed 14
- 15. Sample & AssayTechnologies QIAGEN solution NGS workflow Primary sample to purified DNA DNA preprocessing Target enrichment Library preparation 1. Accessing single cell genomes forfor next generation sequencing Accessing single cell genomes next generation sequencing 2. Target enrichment enabling rare mutation detection and QC 3. Streamlined one-tube library preparation 4. Raw reads to completely annotated variants Sequencing preparation NGS run Data alignment / analysis 15
- 16. Sample & AssayTechnologies REPLI-g Single Cell Kit NGS workflow Primary sample to purified DNA DNA / RNA preprocessing Target enrichment Library preparation Problem: Isolation of NGS compatible DNA quantity from single to few cells Solution: REPLI-g Single Cell Kit – Unbiased amplification of ultra-low DNA amount Sequencing preparation NGS run Data alignment / analysis 16
- 17. Sample & AssayTechnologies Technology Multiple displacement based whole gemome amplification Primers anneal to template DNA Phi 29 DNA polymerase moves along the DNA template strand displacing the complementary strand The displaced strand becomes a template for replication Advantages High yield of high-molecular-weight DNA High fidelity amplification – minimal error rate No effect of secondary structure - minimal bias 17
- 18. Sample & AssayTechnologies How REPLI-g Single Cell Kit work Simple three step protocol (Yield: 20-40 µg) 18
- 19. Sample & AssayTechnologies Comparable NGS results with gDNA & amplified single cells REPLI-g Single Cell technology provide high genome coverage & high fidelity 19
- 20. Sample & AssayTechnologies Unbiased amplification with REPLI-g Single Cell Kit No allele drop out after REPLI-g Single Cell WGA 20
- 21. Sample & AssayTechnologies QIAGEN solution NGS workflow Primary sample to purified DNA DNA preprocessing Target enrichment Library preparation 1. Accessing single cell genomes for next generation sequencing 2. Target enrichment enabling rare mutation detection and QC 3. Streamlined one-tube library preparation 4. Raw reads to completely annotated variants Sequencing preparation NGS run Data alignment / analysis 21
- 22. Sample & AssayTechnologies NGS: DNA sequencing Gene Panels Increase in •Cost •Data complexity Exome Decrease in •Deep sequencing •Multiplexing •Reimbursements Genome Gene Panel Sequencing: Efficient, Effective and Economical method 22
- 23. Sample & AssayTechnologies Clinical utility requires targeted analysis Linking genetic variants with biology Title, Location, Date 23
- 24. Sample & AssayTechnologies Shrink the Genome Focus on Genes of interest relevant to research topic Sample Information Analysis Time Coverage Depth Sample Size Genome 3 x 109 bps 7 days 10X 1 ug 20 Genes 6 x 104 bps 8 hours 1000X 10 ng 24
- 25. Sample & AssayTechnologies Hybridization Which is most suitable target enrichment technology? Hybridization, Ligation + PCR Multiplex PCR 25
- 26. Sample & AssayTechnologies Hybridization Multiplex PCR technology Hybridization, Ligation + PCR Multiplex PCR Workflow: Workflow: Workflow: (SIMPLE) Library construction(4 hrs) Hybridization with probe (16 hrs) PCR amplification (3 hrs) Hybridization with probes (24-48 hrs) Ligation (1 hr) Library construction (4 hrs) PCR & Indexing (2 hrs) PCR & Indexing (2 hrs) DNA input: 1-3 ug DNA input: 200-400 ng DNA input: (LOW) <100 ng Time from DNA sample to NGS library: 2-3 days Time from DNA sample to NGS library: 2 days Time from DNA sample to NGS library: 1 days (RAPID TAT) 26
- 27. Sample & AssayTechnologies GeneRead DNAseq Gene Panel Multiplex PCR technology based targeted enrichment for DNA sequencing Cover all human exons (coding region + UTR) Division of gene primers sets into 4 tubes; up to 1500 plex in each tube 27
- 28. Sample & AssayTechnologies GeneRead DNAseq Gene Panel Focus on your Disease of Interest Comprehensive Cancer Panel (124 genes) Cancer-specific Focused Gene Panels (20 genes) Breast cancer Liver cancer Colon Cancer Lung Cancer Gastric cancer Ovarian Cancer Leukemia Prostate Cancer How Genes on Panels Are Selected Genes Involved in Disease Clinically/Biologically relevant Multiple Publically accessible databases Text mining tools Manually curated Genes with High Relevance Technically relevant Most frequently mutated genes Specific feedback from the thought leaders 28
- 29. Sample & AssayTechnologies Breast Cancer Gene Panel Somatic: All Germline and Somatic: APC, BRCA1, BRAC2, TP53 With approved FDA inhibitor: BRAF, EGFR, ERBB2 29
- 30. Sample & AssayTechnologies GeneRead DNAseq Gene Panel Advantages of Focused and Curated Gene Panel Deep sequencing enabling detection of low prevalence mutations Ion Torrent 316 Chip Competitor A CCP (400 genes) Median Coverage Depth GeneRead Lung Cancer Panel (20 genes) 87X 39.0% Region with >100X coverage 944X 91.2% Multiplex more samples (cost saving) Coverage Depth Competitor A CCP (400 genes) 1000X GeneRead Lung Cancer Panel (20 genes) 1000X Number of Samples 1 8 MiSeq Without missing potentially important mutations COSMIC database Lung cancer specific mutations Competitor A CCP (400 genes) 79.9% GeneRead Lung Cancer Panel (20 genes) 71.4% 30
- 31. Sample & AssayTechnologies Gene Read DNASeq Gene Panel >10 years of proven expertise in primer design: GeneRead algorithm Simple Any gene Any sequencer Any sample: average amplicon size is 145 bp Superior Wet bench verified gene panels – High design rate: >90% – High specificity: >85% – High uniformity (0.1X of median coverage depth): >85% 31
- 32. Sample & AssayTechnologies GeneRead DNAseq Custom Panel 32
- 33. GeneRead DNAseq GenePanel Simple Protocol GeneRead DNAseq Gene Panel PCR primer mix + control primers Add GeneRead Mastermix and genomic DNA (20 ng/rxn) 2 hours Sample & Assay Technologies PCR amplification Pool reactions for each sample and purify (QIAquick PCR Purification Kit) 33
- 34. Sample & AssayTechnologies GeneRead DNASeq Library Quant Array Serial dilution of DNA standard with primer assays for library quantification primer assays for library QC primer assays for Library Quantification 34
- 35. Sample & AssayTechnologies GeneRead DNASeq Library Quant Array Quantification and QC in a single PCR run GeneRead DNAseq Library Quant Array QC Score 1-4 4-8 >8 Results Pass Marginal Fail Proceed Caution! Do not proceed 35
- 36. Sample & AssayTechnologies GeneRead DNASeq Library Quant Array An Example Segregate bad libraries before sequencing; save time and money 36
- 37. Sample & AssayTechnologies DNAseq Panels compatible with NGS platforms No manipulation of target enrichment protocols needed Illumina • • • • • • MiSeq GA Iix HiSeq 1000 HiSeq 2000 HiSeq 1500 HiSeq 100 Ion • • Torrent PGM • 314 • 316 • 318 Proton
- 38. Sample & AssayTechnologies QIAGEN solution NGS workflow Primary sample to purified DNA DNA preprocessing Target enrichment Library preparation 1. Accessing single cell genomes for next generation sequencing 2. Target enrichment enabling rare mutation detection and QC 3. Streamlined one-tube library preparation 4. Raw reads to completely annotated variants Sequencing preparation NGS run Data alignment / analysis 38
- 39. Sample & AssayTechnologies NGS workflow Primary sample to purified DNA GeneRead DNA Library Prep Kits Library preparation Problem Inefficient, complex and error-prone workflow DNA preprocessing Target enrichment Solution Combine various enzymatics steps in single tube Library preparation Fast procedure that allows up to 50% time savings High yields from minimal amounts of starting material Sequencing preparation Unbiased and high-fidelity amplification mastermix (optional) Can be automated on QIAcube NGS run For Illumina & Ion Torrent platforms Data alignment / analysis 39
- 40. Sample & AssayTechnologies GeneRead DNA Library Prep Kits Faster, less variable & more efficient workflow 40
- 41. Sample & AssayTechnologies GeneRead Size Selection Kit Remove DNA fragments <150 bp Before size selection GeneRead size selection Adapterdimers Adaptermonomers Precise size selection of DNA fragments Fast procedure, based on QIAGEN’s proven silica-column technology An easy-to-follow protocol, automatable on the QIAcube 41
- 42. Sample & AssayTechnologies GeneRead DNA Library Prep Kits Input DNA as little as 50 ng High yield of Library DNA Uniform coverage distribution 42
- 43. Sample & AssayTechnologies HiFi Polymerase Comparison: Experimental Design 43
- 44. Sample & AssayTechnologies GeneRead Library Prep Kit Low error rate with minimal sequence bias High-fidelity amplification Greater and more even coverage in GC- and AT- rich areas of DNA 44
- 45. Sample & AssayTechnologies QIAGEN solution NGS workflow Primary sample to purified DNA DNA preprocessing Target enrichment Library preparation 1. Accessing single cell genomes for next generation sequencing 2. Target enrichment enabling rare mutation detection and QC 3. Streamlined one-tube library preparation 4. Raw reads to completely annotated variants Sequencing preparation NGS run Data alignment / analysis 45
- 46. Sample & AssayTechnologies Data Analysis: Free, Complete and Easy to Use 46
- 47. Sample & AssayTechnologies Results in Multiple Analysis Format Run Summary Specificity Coverage Uniformity Numbers of SNPs and Indels Summary By Gene Specificity Coverage Uniformity # of SNPs and Indels 47
- 48. Sample & AssayTechnologies SNP detection Indel detection Features of Variant Report Predicted amino acid change dbSNP and COSMIC ID Effect of SNP Impact of SNP Link to qPCR somatic mutation assay 48
- 49. Sample & AssayTechnologies Genetic variant analysis in FFPE lung adenocarcinoma samples Experimental Design gDNA isolated from 3 FFPE lung adenocarcinoma and one FFPE normal lung samples GeneRead Lung Cancer Gene Panel was used to enrich 20 genes Library preparation, quantification and sequencing QIAGEN NGS Data Analysis Web Portal 49
- 50. Sample & AssayTechnologies Genetic variant analysis in FFPE lung adenocarcinoma samples Run Summary Sample Total number of reads Tumor Sample-1 1,629,983 Tumor Sample-1 1,892,621 Tumor Sample-1 1,851,514 Normal Sample 1,625,119 Specificity (Reads on target) 92% 92% 90% 90% Median Coverage 608 744 703 577 Uniformity (regions with >10% of median coverage) 93% 92% 90% 93% No. of SNPs 84 72 87 83 No. of Indels 8 11 9 6 50
- 51. Sample & AssayTechnologies Genetic variant analysis in FFPE lung adenocarcinoma samples Snap shot of variant report 51
- 52. Sample & AssayTechnologies Streamlined Sample-to-Result Workflow PyroMark Assays 52
- 53. Sample & AssayTechnologies Genetic variant analysis in FFPE lung adenocarcinoma samples Experimental Design gDNA isolated from 3 FFPE lung adenocarcinoma and one FFPE normal lung samples GeneRead Lung Cancer Gene Panel was used to enrich 20 genes Library preparation, quantification and sequencing QIAGEN NGS Data Analysis Web Portal 53
- 54. Sample & AssayTechnologies Genetic variant analysis in FFPE lung adenocarcinoma samples Run Summary Sample Total number of reads Tumor Sample-1 1,629,983 Tumor Sample-1 1,892,621 Tumor Sample-1 1,851,514 Normal Sample 1,625,119 Specificity (Reads on target) 92% 92% 90% 90% Median Coverage 608 744 703 577 Uniformity (regions with >10% of median coverage) 93% 92% 90% 93% No. of SNPs 84 72 87 83 No. of Indels 8 11 9 6 54
- 55. Sample & AssayTechnologies Genetic variant analysis in FFPE lung adenocarcinoma samples Variant Filtering 55
- 56. Sample & AssayTechnologies Genetic variant analysis in FFPE lung adenocarcinoma samples Detection of low frequency variant in lung adenocarcinoma 56
- 57. Sample & AssayTechnologies Data Validation: PyroMark Assay Validation of KRAS:G12V somatic mutation by pyrosequencing assay Tumor sample-1 (KRAS c.35G>T 35%) Normal sample-1 (KRAS c.35G>T 0%) Mutation analysis of codons 12 of KRAS using PyroMark Q24. Upper Pyrogram show G to T mutation in position 1 of codon 12 of KRAS in lung adenocarcinoma sample. The mutation rate (35%) is similar to the NGS results (38%) confirming the reliability of GeneRead DNAseq Gene Panel. The lower Pyrogram shows normal genotype in normal sample-1. 57
- 58. Sample & AssayTechnologies Streamlined sample-to-result workflow PyroMark Assays Gene Reader Eco Solution 2013 Streamlined, standardized and automated sample-to-result workflow 58